Despite intensive treatment that generally combines surgery, radiation, and chemotherapy, oral squamous cell carcinomas (OSCCs) have a long-term survival rate of only 15-50%. Thus, there is a great need for improvements in pharmacologic treatments/chemotherapeutics for OSCCs. One current theory is that conventional treatment fails because it does not adequately treat cancer-initiating cells (CICs), also called cancer stem cells (CSCs). Our laboratory developed the 4-NQO (4-nitroquinoline oxide) carcinogenesis model of oral cancer for mice, now the most widely used murine model for the study of the development of OSCC. When we provide 4-NQO, a carcinogen and a surrogate for the neoplastic lesions caused by smoking, in the drinking water, mice develop lesions in their oral cavities that mimic those in humans, including hyperplasia, dysplasia, leukoplakia, papilloma, and invasive squamous cell carcinomas (SCCs); moreover, the molecular markers of OSCC in this murine model are the same as many of those in human OSCCs. Here we propose to use a cell lineage-tracing approach in this 4-NQO oral carcinogenesis model to test the hypothesis that certain Polycomb proteins which are involved in chromatin regulation, specifically Bmi1, also play a major role in putative CICs in the oral cavity. Bmi1-expressing cells will be permanently marked at the time of tamoxifen addition by using transgenic mice that have a tamoxifen-regulated, creER(TAM) fusion protein gene driven by the Bmi1 promoter, and crossing them with Rosa26 confetti reporter transgenic mice. These mice will be followed during the carcinogenesis process to determine the Bmi1-expressing cell progeny and the expression of Bmi1+ marked cells in OSCCs that develop over time. We will also characterize the functions of Bmi1 in OSCC by over- expressing Bmi1, specifically in the oral cavity epithelium and in a regulated manner, through the use of a doxycycline-regulated expression vector in mice during oral cavity carcinogenesis. Completion of these aims will provide us with much new information about the Bmi1 gene, which is thought to be a key gene required for formation of CICs in human OSCCs. Moreover, the techniques used and further developed in this proposed research will provide us with useful, powerful tools with which to identify and study CICs in OSCC, including their ability to self-renew, their abilityto differentiate, and their phenotypic fluidity. This knowledge is essential to discover new therapies and to screen for drugs that target CICs in human OSCCs.